Sung-Woo Kim is a basic scientist who has been working on the molecular regulation of thyroid I hormone action in the Thyroid Division of the Brigham and Women's Hospital, Boston for the past 6 years. His long-term goal is to define how thyroid hormone negatively regulates multiple genes to generate its effects in various tissues. The hypothesis of this proposal is that important new insights into the mechanisms of negative regulation by thyroid hormone can be obtained by characterization of further examples of negatively T3-responsive genes identified in a rat pituitary cell line (GC). To date, only a few such genes have been studied and there is disagreement about the mechanism. The PI has recently generated about 30,000 15 by cDNA tags from hypothyroid, euthyroid and hyperthyroid GC cells using the serial analysis of gene expression (SAGE) technique. These cDNA pools consist of both known and unknown genes and they have been further classified into those, which are regulated either positively or negatively by T3 at a pretranslational level. Specific Aim I is to identify four cDNAs in GC cells, which are negatively regulated by T3. Two of these will be negatively regulated primarily from the hypothyroid to the euthyroid state and two over the euthyroid to hyperthyroid transition. The cDNAs identified from the SAGE ditags have been used to identify the full length CDNAs of these genes in GenBank. Northern or microarray analyses of GC cells appropriately treated will be used to confirm that the selected genes are directly transcriptionally regulated in these cells. Genes meeting these criteria will be examined in normal, hypothyroid and hyperthyroid rats to allow selection of the most ubiquitously responsive examples. In Specific Aim II, the promoters and 5' flanking regions of these genes will be obtained or isolated. Analysis of the mechanism of action of T3 to cause negative regulation will then be performed using approaches our laboratory and other investigators have successfully employed.